Regulation of the L-type calcium channel

TIPS - October 1987 [Vol. 81 393

39-a 6 Haase, H. J. and Janssen, P. A. J. (1985)

The Action of Neuroleptic Drugs, pp. 123- 222, Elsevier

7 Herrnstein, R. J. (1970) J. Exp. Anal. Behav. 13,243-266

8 Fielding,. S. and Lal, H. (1978) in Neurofeptics alrd Schizophrenia Vol. 10 ([verse& L. L., Iversen, ‘% D. and Snyder; S. H., eds), pp. 91-128, Plenum Press

9 Fischman, M. W. and Schuster, C. R. (1979) Psychopharmacology 66, 3-11

10 Fowler, S. C., Gramling, S. E. and Liao, R. (1986) Pharmaco:. Bioc!rem. Behav. 25, 615-622

11 Heyman, G. M. and Monaghan, M. M. (1987) J. Exp. Psychol. Anim. Behav.

Process 13, 384-394 12 de Villiers. P. A. and Hermstein. R. 1.

(1976) Psychol. Bull. 83,1121-1153 13 Heyman, G. M., Kinzie, D. L. and

Seiben, L. S. (1986) Psychopharmacology Bt3,34&353

14 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. USA 51,660-672

15 Heyman, G. M. (1983) J. Exp. Anal. Behav. 40,113-122

16 Gallistel, C. R. and Karras, D. (1984) Pharmacol. Biochem. Behav. 20.73-7

17 Ham&an, A. L., Stellar, J. R. and Hart, E. B. (1985) Phusicl. Behav. 35,Z97-904

18 Morley, J; M.: Bradshaw, 6. M. and Szabadi, E. (1984) Psychopharmacology 84,444-450

19 McSweeney, F. K. (1978) Anim. Learn. Behav. 6,444-450

20 Lehninger, A. L. (1977) Biochemistry (2nd edn), pp. 189-195, Worth Pub- lishers

21 Clark, A. J. (1933) The Mode of Action of Drugs on Cells, Edward Arnold and Company

22 Heyman, G. M. in Biolopical Determi- nants of Reinforcement and Memory (Church, R. M., Commons, M., Stellar, J. R. and Wagner, A. R., eds), Lawrence Erlbaum (in press)

23 Ayd, F. J.- (1983) in Neuroleptics: Neurochemical, Behavioral, nnd Clinical Perspectives (Coyle, J. T. and Enna, S. J., eds), Raven Press

Regulation of the L-type calcium channel Franz Hofmann, Wolfgang Nastainczyk, Axe1 Rijhrkasten, Toni Schneider and Manfred Sieber

The voltage-operated L-type calcium channel is regulated by protein phosphoylation and G proteins in a variety of tissues and eukayotes including non-excitable cells. The 165 kDa protein of fhe dihydropyridine receptor from rabbit skeletal muscle contains all the regulatory sites of an L- type calcium channel and the calcium conducting unit. Franz Hofmann and colleagues suggest that the differences in fhe regulation observed in various tissues is caused by the interaction of the large conducting protein with different regulatory proteins of approximaieiy 55 ki3a.

There is hardly a tissue where calcium channels have not been postulated as an important part of the signal transduction mechanism. They have emerged early in evolution and have been found throughout eukaryotes including protozoa, algae, higher plants, fungi and animals. Voltage- operated calcium channels are a key component of all excitable cells which transduce electrical signals into biochemical events. In contrast, receptor-operated calcium channels have not been detected by electrophysiological techniques to date, but recent evidence suggests that excitable and non-excitable cells have calcium channels which are regulated indirectly by hormone receptors.

Hormonal regulation of calcium channeis

Voltage-operated calcium chan-

Franz Hofmann is Professor and Ghan-man. WoJfgang’Nastainczyk is Senior Scientist and Axef Riihrkasten, Toni Schneider and Manfred Sieber are Science Assistants at the Physiolo- gische Chemie, Medizinischen Fakultiit der Universtiit des Saarlandes, D-6650 Homburg- Saar, FRG.

nels have been differentiated into T-(transient), N-(neuronal) and L- (long lasting) channels (see Box). N-channels are apparently present only in neuronal cells whereas T- and L-channels have been identified in most cells tested. The cardiac L-type calcium channel was the first channel known to be modulated by hormones13 (see also Table I). Stimulation of ventricular P1-adrenergic receptors activates CAMP-dependent protein kinase and increases 3 to 4- fold voltage-dependent calcium influx.

Perfusion of isolated myocytes with a variety of compounds and enzymes shows that CAMP kinase phosphorylates L-channels or a protein closely associated with L- channels. Single channel recording suggests that phosphorylation decreases the closed times, increases the open time and decreases about 3-fold the number of blanks, i.e. tracings in which the channel does not open upon depolarization. These changes increase about 4-fold the probability that the channel is open and is available for voltage- dependent opening. About 20%

of the cardiac L-channels open in the absence of CAMP-stimulated phosphoryiation. ChanneI opening cannot be decreased further by perfusion of a single myocyte with the specific kinase inhibitor protein, a large excess of the regulatory subunit of CAMP kinase4 or the catalytic active fragment of protein phosphatase I (Ref. 5). This suggests that CAMP- dependent phosphorylation is not a prerequisite for voltage-dependent channel opening.

In contrast, an absolute depend- ence of channel opening on CAMP-dependent phosphorvla- tion has been observed in- an isolated patch of a L-channel excised from a neurosecretory cell line6. Unlike neuronal L-channels, no mechanism has been identified in cardiac ventricular cells which modulates L-channels without affecting the activity of CAMP- dependent protein kinase.

Phosphorylation of the cardiac L-cha?tncl is stimulated in vivo by all hormones which activate adenylate cyclase. An increased current can be decreased by hormones which inhibit adenylate cyclase activity. Stimulation of ventricular muscarinic receptors decreases the calcium current through the latter mechanism by activation of the inhibitory GTP- binding protein Gin The calcium current is also decreased by cGMP which lowers the CAMP level by activation of a cGMP-stimulated CAMP phosphodiesterase’. Both mechanisms result in a dephos- phorylation of the channel and thereby decrease the open state probability.

Neuronal calcium currents are inhibited by a variety of adrenergic and peptidergic receptors (see Table I). These receptors activate a pertussis toxin-sensitive GTP- binding protein, which couples

@ 1987, Eleevier Publications, Cambridge 0165 - 6147/87/SOZ.W

394 TIPS - October 1987 [Vol. 83

TABLE I. Hormonal regulation of calcium channels

Ttasuedl HoIlllnm, Effeotor current Ref.

and possibly in other tissues. It is possible that IP4 is the true and direct activator of the channel since many cells can phosphory- late IPs to IP4 A release of calcium from intracellular stores is not required for activation of this channel. IPs/IP* may also be the link between angiotensin II receptor activation and the increase in a voltage-dependent calcium current of the murine adrenocortical cell line Y-l cells. The stimulating effect of angiotensin II is pertussis toxin-sensitive.

!zztal

Neurons DBG NG19%15

NA (6th H CAMP kinase increase cAMP kinase increase

NA, DA, 5HT. GABA G protein decrease Gsoiate. ST G, (ar-subunit) decrease

a-d e

f-h ii .

Aplysia PKC new channel k

HefNaspwa cAMP kinase increase I 5-HT cGMP kinase increase m cholecystokinin PKC decrease n

Endocrine cell lines Pitutkary ST G protein decrease 0

CAMP kinase increase P Y-l angiotensin II G, increase q

othercells T I-$yihiyTes iiiiiOgerl.5 IPJIP, increase r Fiibiast PKC increase S

fMLP IP4 ca!dum

increase iiiiXS~SS*

t U

l mnwpscBc cation channel. NAnoradrenaline. H, histamine; DA, dopamine; ST, somatostatin; PKC. protein kinase C; FMLP. fMET’-Leu-Phe.

%euter. H. (1974) J. Physiot. 242,429451; bOsterrieder, W. etai. (1982) fVafure298,576- 573: Karnayama, M. et al. (1986) PrTrigers Arch. 407. 466-463; dHescheler, J. et al. (1986) PR6gers Arch. 497. 182-189: %chmid, A. et al. (1985) J. Biol. Chem. 260, 13041-13046; ‘Rane 6. St. and Dunlop, K. (1966) Proc. NatlAcad. Sci. USA 83,184-188; eHolz, G. G. IV et a/. (1986) Nature 319.670-672; “Marchetti, C. et at. (1966) Pf/-

uig ers Arch. 406, 104-111;

%uno. A. ef al. (1966) Pruc. Natt Acad. sci. USA 63.9832+636; Hescheler. J. et a/. (1987) Nahrre 325, -7; %trong, J. A. et a/. (1987) Nafun? 325, 714-717; ‘Chad, J. E. and EckeR R. (1986) J. physid. 378.31-51: mPaupardin-Trftsch, D. et al. (1986) Nafure 323,812- 814; “Hammond, C. et at. (1987) Nature 325,809-811; OLewis, D. L. et al. (1986) Proc. Nat/ Acad Sci. USA 83.9035-9039; PAnstrong, D. and Eckert. f?. (1987) Proc. Nat/ Acad. Sci. USA 64.2519-2522; qHescheler, J. et al. (1987) Naunyn-Schmied. Arch. Pharmacol. 335, (suppf. B 34): ‘Kuno, M. and Gardner, P. (1987) Nature 326,301-304; aChen, C. and Hess, P. (1987) Biophys. J. 51.226a;%vine, R. F. and Moor, R. M. (1986) Biochem. J. 240.917-920; “Tanabe. T. eta/. Nature 328 (in press).

directly or indirectly to the channel by an apparently CAMP- independent mechanism. The inhibition was reported to be specific for L-channels in NGlOS- 15 cells, whereas in chick dorsal root ganglions inhibition of T- and L-channels has also been observed. An inhibition of N-channels by noradrenaline has been observed in frog sympathetic neurons. The calcium current of NG108-15 cells is decreased by activation of a-opiate and somatostatin receptors. Hormonal inhibition was completely recovered in pertussis toxin-treated cells by injection of the brain-specific GTP-binding protein G, or its o-subunit. It is not clear if the a-subunit of G, couples directly to the channel. An indirect coupling could be mediated by protein kinase C (Ref. 8) although most groups have failed to see an effect of phorbol esters on neuronal calcium channels.

Phosphorylation of neuronal channels has been studied mostly in invertebrates since these neurons are more suitable for injection experiments. Perfusion of helix neurons with CAMP and ATP prevents the time-dependent

inactivation of L-type channels. The injection of cGMP kinase - the first report of an effect of cGMP kinase on a channel - increases whole cell calcium current of specific snail neurons. Injection of protein kinase C or treatment of snail neurons with phorbol esters resulted in cell- dependent effects. In one report, induction of a new calcium channel was seen whereas in another an inhibition of whole cell calcium current was observed. The channel type modulated by hormones in snail neurons has not been tested in each case but most of them are presumably L-type channels. This suggests that hormonal regulation of neuronal calcium channels is more diverse than that of the cardiac L-channel.

This already complicated pic- ture is further modified by recent reports that non-excitable cells have calcium channels which can be regulated by hormones. Acti- vators of protein kinase C increase L-type calcium channel current in mouse 3T3 and human fibroblasts. IPs and IP4 activate directly a plasma membrane localized calcium channel in T lymphocytes

In contrast to the neuron;;! L- channels, the stimulation by angiotensin II was not restored by injection of the GTP-binding pro- tem G, but by Gi. The angiotensin II-stimulated calcium current was blocked by organic calcium channel blockers, suggesting that this channel is related to the L-channel of excitable cells. These channels are different from the non- selective cation channel studied by Reuter and colleagues in fMLP (fMet=Leu-Phe)-stimulated leuco- cytes which opened only after IPs- induced release of calcium from intracellular stores.

Biochemistry of L-calcium channels

The L-type calcium channel has

kDa

55 -- 0

32 - l SW

DTT - +

Fig. 1. Peptide composition of the purified skeletal mu&e dihydropyridine receptor. The receptor was purified (for methodology, see Ref. 151 and denatured in the absence (-) and presence (+) of dithiothreito/ (On). The proteins (300 ng) were separated on a 7.5% SDS-PAGE and stained wkh silver.

TlPS - October 1987 [Vol. 81 395

“PIwe@ different types of voltage-operated calcium channels

Three different types of voltage-operated calcium channels have been defined by electrophysiofogical and ph~acolo~c~ techniques: T-(transient); N-(~~~R~); and L-(long Yasting) channels”.

L- and T-channels have been identified in variable concentwiions in neuronal, cardiac, skeletal, smooth muscle and neurosecretory &Is. T-channels - concurrently used names are low voltage activated (L.VA)*>, and fast inactivating (fast) channel - conduct calcium equally, if not better than barium, whereas L-channels - concurrently used names are high voltage activated W’JA)ls, and non- or slow inactivating channels - conduct barium better than calcium. N-channels have %een described or,‘; for rSzr#ns*.

T-channels are not affected by the known calcium channel agonists, antagonists or toxins. They we inhibited by 0.1 rnM nicke14._ and in c>zrtain neurons by 0.1 rnM phenytoin3. N- and L-chamtels are blocked by low concentrations of cadmium ;ZOw)‘. N-channels and neuronal but not cardiac, skeletal and smooth muscle L-channels are blocked by o-conotoxin5 suggesting differences between neurunal and non-neuronal L-channels. Apamin, a blocker of calcium-dependent potassium channels, apparently blocks cardiac L-channel@.

Channel txmductem# Agonist Antagonfst

T f-apsi nickei (0.1 mM) N 13 psi cadmium (20 HAM)

w-conoioxfn L 2&-25 p% atrotoxin cadmium (20 pa)

maitotoxin ol-conotoxinb goniopora toxin apamirP (+)-20 2791 1,4-dihydropyridines BayK9944 phenyfalfcyfamines CGP 29392 ~~o~i~~e

“Unitary current measured with 90-l 10 mM BaCI,. b Reported to block onlv vertebrate neuronal L-channels. ‘Reported to block cardiac t .-channefs. (+)-29 2791; (+)~~~yl4-(2,1 ,bbsnzoxa- diazol+f)-l~4-~h~m-2~~m~yl-~n~ro-3-~ddine carboxylate]. r2GP 28392; 4-[2--(diffuormethoxy)phenyblh5,7-tetrahydro- 2~~m~~hyl-!%oxofuro[3,4-b]pyrfdine-3-carboxycylic acid ethylester.

The peripheral and neuronal L-channels are activated by maitotoxir?B, atrotoxing, goniopora toxi#’ and the IA djhy~~~~e, Bay K S6M1”**, CGP 28392” and (+)-2027!91 .

Neuronal and peripheral L-rhannels are also the targets for the organic calcium channel blockers which fall into three classes: the 1,4~y~~~, the phenylalkylamines and the benzothiazepines14. With the exception of verapami13, these compounds appear to be specific for L-channeIs if used at low to moderate concentrations but affect a variety of other membrane systems if used at hi&er concentrations. A preference of some compounds for central v. peripheral L-&annd has been noted. These pharmacological data support the notion that neuronal and peripheral L-chmels may differ considerably.

Referenee8 I Carbone, E. and Lux, H. D. (1984) Nutare 310,501-503 2 Nowycky, M. C., Fox, A. P. and Tsien, R. W. (1985) Nature

316,44&443 3 Yaari, Y., Hamon, B. and Lux, H. D. fl9tI7) Scimce 23S, 6gCt-

682 4 Fedulova, S. A., Kostyuk, P. G. and Veselovsky, N. S. (1985)

J. PhysioJ. &md.) 359,431-446 5 Cruz, L. J., Johnson, 0. S. and Olivera, B. M. (1987) Bio-

chemistry 26,820-824 6 Bkaily, Gh., Speretakts, N., Renaud, J-F. and Payet” M. D.

(19c)5) Am. J. Pkysiol. 248, H961-H965 7 Takahashi, M., Tatsumi. M. and Ohimmi, Y. (19g3) I. f&f.

Chem. 258,10944-10949 8 Friedmann, S. R., Miler, R. J., Miler, D. M. and Tindali,

D. M. (1984) Proc. J&H Acud. Sci. USA al, 458243% 9 Hamilton, S. L.. Yatani, A., Hawkes, M. J. and Bmwn, A. M.

(1985f Science 229,182-184 10 Qar, J., Schweitz, H., S&mid, A. and Lazdunski, M. (1986)

FEBS L&l 202,331-336 11 Kokubun, S. &td Reuter, H. (1984) Prec. Natt Acad. Sci. USA

81,4824-8827 12 Nowvckv, M. C., Fox, A. P. and Tsien, R. W. (1985) Proc.

N&J k&i. Sci. UsA 82,2178-2182 13 Hof, R. P., Riiegg, V. T, Hof, A. and Vogel, A. (19Bs)

J, Cardiovasc. Pkannacof. 7,689-693 14 Triggle, D. J. and Janis, R. A. (198’7) Annu. Rev. Phannacol.

Toxical. 27,347-390

been identified in vitro by its three drug binding sites which are specific for dihydropyridines, phenylalk~lamines and benzothia- zepinesg- l. These stereospecific sites have a high affinity for the respective group. The binding to one site is regulated allosterically by the occupation of the other sites, by divalent cation and by temperature. Most tissues contain in addition to these high affinity, allosterically regulated sites, low affinity, high capacity binding sites for calcium channel blockers, which are not located on the L-channels”‘. In agreement with electrophysiological data, the density of the high affinity, allosterically regulated sites is low in cardiac muscle and other tissues. The only exception is the trans- verse tubulus of skeletal muscle

which contains a high density of these sites, i.e. 10 to 80 pmol per mg T-tubulus protein13.

The high affinity dihydropyridine receptor purified by a three- step procedure from these membranesI contains three proteins of apparent molecular weight X65,55 and 32 kDa (Fig. 1). These three proteins are purified in a constant ratio of 1:1.7:1.4 suggesting that they may belong to the same molecular structure15. The purified receptor binds all three major classes of calcium channel blockers15-‘8 , i.e. dihydropyricImes, phenylalkylamines and diltiazem, in a stereospecific manner (Table II). Binding to the dihydropy~dine site is regulated temperature- dependently and allosterically by (-)-desmethox~erapamil, ver- apamil and (+)-cis-diItiazem’6.

Equilibrium binding experiments with (+)-PN 2@0-110 [isopropyl 4-(2,1,3,benzodiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxycar- bonylpyridridine-3-carboxylate] indicate that the receptor is about 30% pure.

The proteins of the purified receptor can be separated on a gel filtration column in the presence of SDS (Fig. 2). The photoaffinity ligand for the dihydrop~dine site, azidopine”, and that for the phenylalkylamine site, Lu 49888, [(A)-5-(zaziclophenethylmethyl- amino)-2-(3~~~-metfioxyphenyi)- 2-isopropyl-valeronitril] specifically label the 165 kDa protein’5*‘9*20 suggesting that this peptide contains the high affinity sites for the calcium channel blockers (Fig. 2). The 165 kDa peptide is phosphorylated by CAMP-dependent protein

3%

TABLE Il. pmpertios of the @lied c&&m channel from r&bit sketetai mu&e

P--lcomposltfon 165,55,32 kDA Cal&m channel blocker receptor 185 kDa protein Calcium channel conducting unit 165 kDa protein CAMP k&~ substrate (1.8 mot mot-‘) i 65 kDa protein ?w~eti~ wasa C substrate (1.0 mot mar7 55 kDa protein

~~~,~) & 5-10 rw 1 site

~~~) ~~~~~1 site 50

Al- mguWon and stemospac%c binding

a- =20 psi Wtaga&psndant regulation yes (bell-shaped) phannacologicai regulation Yes ~~~~~ yes

W88, 2,?~~yi~(3.4~ime\hoxyphenyt)-39yan-?~a~Q-(3-metho~phenyl)n~an~

kinase up to a stoichiometry of 1.8 mol phosphate per moi of the X5 kDa protein. CAMP kinase incor- porates in less than i0 min one mol phosphate per mol 165 kDa protein, supporting the physiological importance of this phosphorylation; CAMP kinase also phos- .phorylates the 55 kDa protein up to one mot phosphate per mol protein. Comparison of the phosphorylation rates of various protein kinases shows that the 55 kDa protein is a preferred substrate for pm&in kinase C (Ref. 21 aud Fig. 3). These results show that the 165 kDa peptide contains all the regula.ory sites known to affect the L- type calcium channel in &ZID, i.e. sites for organic calcium channel blockers, activators and CAMP- dependent phosphorylation. The nature of the 55 kDa and 32 kDa protein is unclear. Peptide maps of each protein indicate that they are unrelated to each other’s.

The purified dihydropyridine receptor is contaminated to a variable degree with another protein which has in the absence of reducing conditions an apparent molecular weight of 165 kDa, but yields two peptides of 130 and 28 kDa in the agents15,16z

presence of reducing (see also Figs 1 and 2).

A 130128 kDa protein has been purified by others as a calcium channelas. This pmtein, but not the 165 kDa receptor protein, is a glycoprotein22. Antibodies against it stain the same protein in skeletal, cardiac and smooth musclez6 and the same or a similar protein has been purified from chick heart”. Recently# it was suggested that it is derived by limited proteolysisui from the 165 to 170 kDa reseptor pmtein which is specifically labelled in T-tubuiar membranes by the dihydropyri-

dine PN200-110, bebridil and (+)- cis-diltiazem20 although these results have not been confirmed by otber~~~~~. V-8 protease peptide maps for the 165 kDa receptor and the 130/28 kDa protein are unrelated15. In contrast to the known pharmacology of the L- type calcium channel, the 130/28 kDa protein does not bind di- hydmpridines or phenyl~k3lami- nes with high affinity (Fig. 2) and is not phosphorylated by CAMP kinase. It is therefore unlikely that it represents the high affinity receptor for calcium channel blockers which is a part of the L- type calcium channel.

Purification of the high affinity dihydropyridine binding receptor from bovine cardiac muscle yields three peptides with apparent molecular weights of 183,172 and 110 kDa. The photoaffini~ ana- logs aridopine and Lu 49888 are incorporated only into the 183 kDa peptide. Neither the two smaller proteins nor a 56 or 36 kDa protein are labelled by these compounds. This suggests that the high affinity binding sites for calcium blockers have an apparent molecular weight of 183 kDa in cardiac muscle and of 165 kDa in skeletal muscle. A very similar molecular weight has been determined for the membrane bound receptor by specific antibodiesz6.

Reconstitution of L-type channel The purified dihydropyridine

receptor from skeletal muscle con- taining the 165, 55 and 32 kDa proteins has been reconsti~ted into phospholipid vesicles con- taining phosphatidyl ethanol- amine, phosphatidyl serine and cholesteroi2r. These vesicles fuse with a phospholipid bilayer formed at the tip of a patch

TIPS - October 1987 [Vol. 81

pipette. After fusion, spontaneous single channel openings are recorded in symmetrical 90 mM 13aC12 solutions. In agreement with whole cell recording data, the reconstituted protein channel has a single channel conductance of 20 psi. Its open state probabili~ (P,) is reduced by the calcium channel blocker, gallopamil and PN 20% 1.10 and increases in the presence of calcium channel agonist Bay K 8644 [methyl 1,4-dihydro-2,6- dimethyl3nitro - 4 - (2 - trifluoro- methylphenyl)-p~d~e-5~~b~ late]. The P, is also increased several fold by the a&lition of ATP-magnesium complex, in<.!. 2he

I Gel-fil~ation

60 80 100

kDa

2UJ- 116-

g-

I II III IV

SDS-Gel I8R”

-I

x10-3 3

$ 0 2 .g ‘6 a, w

1

Retention time (mint

Fig. 2. Separation of the pepfides of the purtfied dihydtvpytdine receptor from skeletal muscle For each exoer- iment 35 pg of p&&d receptor biers denatured in the oresence of SDS and separated on 6 TSK-G l3OCkMOgU cotumn system (sse fief. rs). The top panel shows ths absorbance at 280 nm f--f. The hatched fractions were boilid ‘in the a&ence (-) and presence f+) of 2 mM D7Tand separated on a &% SDS-PAGE (middle’panst). The towerpanelshows the distribution of the spe&catfy incwporated piWo- a~~~~~~~~~(o)~d (%t]Lu 48999 (0) after UPLC-get f&a- tion. Each fine is the di&ence of two separate expsttments in which the tdtiated @and was photolysed In the absence and presence of a thousand. fold excess of unlabeled tigand.

TlPS - October 1987 [Vol. 81

catalytic subunit of CAMP-dependent 7protein kinase. These results* suggest that the reconstituted channel has many pro- perties of the cardiac L-type calcium channel.

Further analysis of the single channel kinetics*s showed that open and closed times of the reconstituted channel are about 10 times longer than that of an in-viva or in-vitro2g,31 measured cardiac muscle L-type channel, indicating that the purified and reconstituted skeletal muscle channel has prop- erties which differ from that of the cardiac muscle channel. The slower channel kinetics of the purified receptor are not caused by proteolysis of the receptor dur- ing purification. The same kinetics were obtained when solubil- ized microsomal membranes or T-tubular membranes were reconstituted.

Both preparations, the solubil- ized membranes from skeletal and cardiac muscle- and the purified skeletal muscle receptor, contain a second channel with a conductance of 8.8 pSi28*31-33. The P, of the channel with the smaller conductance was not afiected by phosphorylation, calcium channel blockers or agonists, but its open state probability was regulated by volta e

P similar to an in-vivo

channel* . The large conductance yielded a bell-shaped voltage dependency. P, was greatest at a membrane potential around 0 mV and decreased when negative or positive membrane potentials were applied28. These differences in electrophysiological parameters clearly distinguish the two conductances and suggest that they could represent the recently identified ‘fast’ and ‘slow’ calcium conductance of skeletal muscle T-tubuh#. However, other groups35*J6 have reconstituted crude and purified skeletal muscle calcium channels which showed several conductance sub-states ranging from 3 to 12 psi and 4 to 50 psi in symmetrical BaClz solutions. These sub-states were sensitive to calcium channel agonists and antagonists. Although these sub-states could be artefacts of the reconstitution procedures, they could also be a property of the native channel since recently an intermediate sub-conductance level has been observed in cell- attached recordings of the cardiac L-type calcium channel.

kDa PKC PKA 165- m-0 -w

--m l = m

5 10 15 20 40 80 120 5 10 15 2040 80 120

Minutes

Fig. 3. Phosphorylation of the purtfied dihydropyridine receptor. The purified receptor (1.6 pg) was phosphotylated in the presence of 1.25 nM protein ktnase C (PKC) or2 nM CAMP kinase (PM) (for methodotogy see Ref. 27). The radioactivity incorporated into proteins was determined after electrophoresis by autorad@vaphy. PKC (60 kDa) and a contaminating 40 kDa protein are phosphorytated also (leti panel).

The biochemical identity of the two conductances or channels is not clear at present. Recent experiments with the isolated 165 kDa receptor protein show that the large calcium conductance can be reconstitllted by the 165 kDa protein alone. This interpretation is supported by the recent cloning of the 165 kDa receptor

s rotein from

rabbit skeletal muscle 7. The prim- ary structure of this protein is homologous with that of the voltage-dependent sodium channel and could therefore represent part of a calcium channel. However, the kinetics of the purified reconstituted channel differ considerably from that of cardiac muscle L- type calcium channel suggesting that the skeletal muscle dihydropyridine receptor does not contain the complete structure of a cardiac L-type calcium channel.

0 0 0

Calcium channels which are regulated directly by the binding of a hormone to the channel protein have not been identified so f r. i% -c-r qr-operated channels can I e explained by an indirect regulation of the channel through hormone recent<+dependent activation of speci& GTP binding proteins. These bind either directly to the channel or increase the concentration of lP$lP+ or other second messengers. Stimu- lation of these second messengers may be connected with the activation of Rrote’n kinase C as exem- plified by the protein kinase C stimulated L-type calcium channels of fibroblasts. In addition, the activated G protein may affect the membrane potential by decreas- ing the potassium conductance.

Thus receptor-operated calcium channels may turn out to be a sub- species of L-type calcium channels which, like other L-type channels, are regulated biochemically and by the membrane potential.

The L-type calcium channels present in different vertebrate tissues are not identical. Remark- able differences between cardiac and skeletal muscle channels have been noted. Reconstitution experiments suggest that the 165 kDa dihydropyridine receptor protein of skeletal muscle contains the calcium conducting unit of a L- type calcium channel. In bovine cardiac muscle this unit may be confined to a 183 kDa protein. Both channels are modulated by phosphorylation of the channel protein to increase their open state probability. Smooth muscle and vertebrate neuronal L-channels are different since they are not affected by cAMP-dependent phosphorylation. Furthermore, the conductance through vertebrate neuronal L-channels is decreased by G proteins. This and the different pharmacology suggests that vertebrate neuronal and smooth muscle L-channels are a further sub-species of L-type channels.

The molecular basis for these differences can be derived from the work with skeletal muscle. Vertebrate skeletal T-tubulus contains a dihydro yridine-sensitive voltage senso J-4 1 which may couple membrane excitation with contraction and a calcium channel sensitive to calcium channel blockers. The kinetics of this channel are slower than that of cardiac muscle, in agreement with the slow kinetics of the purified, reconstituted channel. Reconstitu- tion of the purified 165 kDa

TIPS - October 1987 CVol. 81 398

dihydrupyxidine receptor pr&ein in the absence of sknificant amounts af the 55 kQa- protein yields a channel with conductances ranging from 3 to 50 pSi35*a. It is therefore possible that the 165 kRa protein which contains the drug binding sites and the Cat- cium conducting unit is itself insufficient to reconstitute a regu- lar L-type calcium channel.

This interpretation is supported by the finding that the mRNA derived from the cDhJA of the 165 wla receptor-w was not able to induce the expression of calcium channels in oocytes. The sequence of the cloued receptor suggests that the receptor could serve as voltage-sensor mdhr d&m &annel. Therefore, the rqg?dar f3~physioIogy of a trrype cd- cium chmd may depend an the interactiou of a large conducting unit and a smaller regulatory protein. The differences in L-type ca?cium channel are probably caused by the expression of homofopous but distinct calcium pore proteins with molecular weights around 200 kDa and different reguiatory proteins of approximately 50 kDa. Thus, the interaction of a slightly different pore protein with a family of xeguLatory proteins may be the mohxular basis for the differences in calcium channel regulation in different tissues.

We thank Mrs. hIage and k&s HeR for technical assistance, Mrs Poesch, for typing the manuscript and Mrs Siepmann for the graphi- cal work. Part of this work was supported by grants from DFG and Fond der Chemischsn Indus- trie.

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A program for the Megraked J. W* Black, D. H. ~~~~~~~ and v. M~~ae~~-Men~n equation, Zzy P. ~~~ku~~~~~, Alan l&s 7987, G. Tkwnas, f-C. ~fflabard and C. &$7.i?O (xi f 2951 iSBN 0 8451 Give (August 1987, pp. 292-294) 3705 0. In the first paragraph describing the integrated Michaelis-Menten equation the correct version of

Synthetic inhibitors of human

the equation should be: neutraphU elastase, by Diane Amy Trainor (hqpsf X93?, pp.

C(t) - c, + &LIT@)] -!- %‘max f = 0 303-3071. 0 The correct structure of the tri-

A step nearer classificatian ffuoromethyl ketones is as fallows:

(book review/August 1987, pp. 320-321) In the book, Perspectives on Receptor Classification: Recep- tor Biochemistry and Method-

~~~~~_~~

ology Vol. 6 the correct version of editors should read, edited by We apologise for these errors.

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Regulation of the L-type calcium channel